The present invention is generally in the field of genetic engineering, and specifically, in the area of manipulation of amino acid biosynthesis in Gram positive bacteria.
Corynebacterium glutamicum is a Gram positive, nonpathogenic microorganism that has long occupied a central role in the industrial production of amino acids by conventional fermentation processes. Past strain development has primarily depended on classical mutagenesis to remove competing pathways to thereby increase substrate availability, and to remove or reduce regulatory control of a particular biosynthetic pathway. Regulatory mutants were isolated by selecting strains resistant to toxic amino acid analogues. The use of chemical mutagenesis has been very successful and a number of economically viable L-amino acid fermentation strains, such as strain producing L-glutamate and L-lysine, have been established.
The recent development of cloning vectors, including those described in U.S. Pat. No. 4,649,119 to Sinskey et al., and methods for DNA transformation of C. glutamicum, as decribed by Katsumata et al., J. Bacteriol. 159, 306-311 (1984), and Yoshihama et al., J. Bacteriol. 162, 591-597 (1985), and the closely related Corynebacterium (Brevibacterium) lactofermentum described by Santamaria et al. in J. Gen. Microbiol. 130, 2237-2246 (1984), initiated a new era in the genetic manipulation of these organisms.
However, the commercial utilization of C. glutamicum recombinant DNA technologies for future strain development is dependent on the development of additional genetic tools and a better understanding of the fundamental molecular biology of this species. The use of recombinant DNA techniques to develop industrial strains would offer several advantages over classical mutagenic strategies. For example, specific alterations such as the replacement of a low efficiency promoter would be possible, the stepwise isolation of enhancing mutations could be avoided, regulatory systems could be engineered to allow the temporal control of gene expression during a fermentation process, and novel genes and/or pathways could be introduced into an organism.
It is therefore an object of the present invention to isolate and characterize genes encoding components of amino acid biosynthetic pathways in Corynebacterium.
It is another object of the present invention to clone the isolated amino acid biosynthetic genes, specifically those involved in the threonine biosynthetic pathway.
It is still another object of the present invention to elucidate the structure of these genes and the regulatory mechanisms that modulate their expression.
It is a further object of the present invention to characterize and modify the expression of the cloned, amino acid biosynthetic genes, as well as the primary structure and regulatory features of their protein products.